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Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin

Current-voltage curves for DIDS-insensitive Cl(−) conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellula...

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Autores principales: Freedman, Jeffrey C., Novak, Terri S.
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1997
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2220065/
https://www.ncbi.nlm.nih.gov/pubmed/9041449
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author Freedman, Jeffrey C.
Novak, Terri S.
author_facet Freedman, Jeffrey C.
Novak, Terri S.
author_sort Freedman, Jeffrey C.
collection PubMed
description Current-voltage curves for DIDS-insensitive Cl(−) conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl(−) conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K(+) and Cl(−) were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl(−) effluxes, induced by valinomycin or gramicidin at varied [K(+)](o), were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K(+)](o). Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P(K)/P(Cl) is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P(Cl). Fitting the constant field current-voltage equation to the measured Cl(−) currents yields P (Cl) = 0.13 h(−1) with DIDS, compared to 0.49 h(−1) without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl(−) current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3.
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spelling pubmed-22200652008-04-22 Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin Freedman, Jeffrey C. Novak, Terri S. J Gen Physiol Article Current-voltage curves for DIDS-insensitive Cl(−) conductance have been determined in human red blood cells from five donors. Currents were estimated from the rate of cell shrinkage using flow cytometry and differential laser light scattering. Membrane potentials were estimated from the extracellular pH of unbuffered suspensions using the proton ionophore FCCP. The width of the Gaussian distribution of cell volumes remained invariant during cell shrinkage, indicating a homogeneous Cl(−) conductance among the cells. After pretreatment for 30 min with DIDS, net effluxes of K(+) and Cl(−) were induced by valinomycin and were measured in the continued presence of DIDS; inhibition was maximal at ∼65% above 1 μM DIDS at both 25°C and 37°C. The nonlinear current-voltage curves for DIDS-insensitive net Cl(−) effluxes, induced by valinomycin or gramicidin at varied [K(+)](o), were compared with predictions based on (1) the theory of electrodiffusion, (2) a single barrier model, (3) single occupancy, multiple barrier models, and (4) a voltage-gated mechanism. Electrodiffusion precisely describes the relationship between the measured transmembrane voltage and [K(+)](o). Under our experimental conditions (pH 7.5, 23°C, 1–3 μM valinomycin or 60 ng/ml gramicidin, 1.2% hematocrit), the constant field permeability ratio P(K)/P(Cl) is 74 ± 9 with 10 μM DIDS, corresponding to 73% inhibition of P(Cl). Fitting the constant field current-voltage equation to the measured Cl(−) currents yields P (Cl) = 0.13 h(−1) with DIDS, compared to 0.49 h(−1) without DIDS, in good agreement with most previous studies. The inward rectifying DIDS-insensitive Cl(−) current, however, is inconsistent with electrodiffusion and with certain single-occupancy multiple barrier models. The data are well described either by a single barrier located near the center of the transmembrane electric field, or, alternatively, by a voltage-gated channel mechanism according to which the maximal conductance is 0.055 ± 0.005 S/g Hb, half the channels are open at −27 ± 2 mV, and the equivalent gating charge is −1.2 ± 0.3. The Rockefeller University Press 1997-02-01 /pmc/articles/PMC2220065/ /pubmed/9041449 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Article
Freedman, Jeffrey C.
Novak, Terri S.
Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
title Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
title_full Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
title_fullStr Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
title_full_unstemmed Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
title_short Electrodiffusion, Barrier, and Gating Analysis of DIDS-insensitive Chloride Conductance in Human Red Blood Cells Treated with Valinomycin or Gramicidin
title_sort electrodiffusion, barrier, and gating analysis of dids-insensitive chloride conductance in human red blood cells treated with valinomycin or gramicidin
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2220065/
https://www.ncbi.nlm.nih.gov/pubmed/9041449
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